Process for producing activated human ALT

ABSTRACT

The present invention relates to an altered-type human ALT gene in which the codons for the five amino acids in the human ALT (alanine aminotransferase) gene are replaced, i. e. the fourth amino acid codon from the initiation codon for methionine (Met) is replaced by a codon for serine (Ser), the fifth by a codon for threonine (Thr), the seventh by a codon for aspartic acid (Asp), the 39th by a codon for glycine (Gly) and the 222nd by a codon for alanine (Ala), and concurrently, restriction sites are added at the upstream and downstream of said gene. 
     The active human ALT having properties similar to those of the native enzyme can be effectively produced by culturing  E. coli  transformed with a recombinant plasmid in which the altered-type human ALT gene of the present invention is ligated to a vector.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to the production of a human ALT (alanine aminotransferase), and particularly, an active human ALT maintaining a sufficient enzyme activity and having a similar property to that of the native enzyme.

More precisely, the present invention relates to a novel gene encoding an amino acid sequence of a human ALT, a novel plasmid having an altered-type human ALT gene having restriction sites added at the upstream and downstream of said gene, and Escherichia coli transformed with said plasmid, as well as a process for production in which the human ALT is expressed as an active enzyme using said Escherichia coli.

DESCRIPTION ON THE RELATED ART

Human ALT is an enzyme that is leaked into serum in liver diseases such as viral hepatitis, hepatic cirrhosis and the like, and is important in clinical chemistry. In the serum diagnosis, standardization such as minimization of the difference between laboratories on measured values of ALT activity and the like is one of important problems. While a crude product originated from porcine heart is presently used as the standard for such purpose, this product is different from human enzyme in enzymological properties such as substrate specificity, Km values and the like, and therefore, there has been a demand for commercialization of an active enzyme originated from human.

On the other hand, the production of a protein derived from a heterologous organism in a microorganism became possible and it has been put to the practical application, thanks to the recent genetic engineering technology. For example, the production of an animal protein in Escherichia coli into which a plasmid formed by ligating a lactose promoter to plasmid pBR 322 is introduced is described in Science, 198, 1056, 1978. While, in the case of human ALT, the gene is cloned and its expression in Escherichia coli is attempted, there has not been an article reporting the expression of the ALT protein maintaining a sufficient activity.

The present inventors have also attempted previously the expression of a recombinant human ALT as an active holoenzyme in Escherichia coli, but fails to successfully obtain a high expression of the desired recombinant enzyme (Japanese Patent Publication (A) Hei 8-103278).

In the field of clinical laboratory test, standardization such as minimization of the difference between laboratories on measured values of ALT activity and the like is one of important problems. While a crude product originated from porcine heart is presently used as the standard for such purpose, this product is different from the human enzyme in enzymological properties such as substrate specificity, Km values and the like. Therefore, there has been a demand for an active enzyme originated from human, but a large supply of the enzyme from human tissues has been difficult.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a DNA sequence of a human ALT gene according to the present invention (SEQ ID NO:1) and its encoded amino acid sequence (SEQ ID NO:2).

FIG. 2 shows a continuation therefrom.

FIG. 3 shows a continuation therefrom.

FIG. 4 shows primers for PCR.

FIG. 5 shows a process for preparing recombinant plasmid pTRAL-F140 of Escherichia coli having a human ALT gene altered by PCR.

FIG. 6 shows recombinant plasmid pTRAL-F140.

FIG. 7 shows expression vector pTRP.

FIG. 8 shows the total nucleotide sequence (SEQ ID NO:14) of the expression vector PTRP.

FIG. 9 shows a continuation therefrom. As the result of base sequencing, it was found that the open reading frame contained only a structural gene for β-lactamase.

DESCRIPTION OF THE INVENTION

As the result of studies made from various directions for solving these problems, the present inventors have again paid attention on the previous research, made by the present inventors, in which the expected result was not sufficiently achieved; have desired to solve the problems by modifying/optimizing the nucleotide sequence of the human ALT gene; have conducted extensive studies on specific measure for such purpose; have succeeded in alteration after repeating trial and error; and have successfully confirmed the expression in Escherichia coli and also confirmed the fact that the obtained altered-type human ALT is excellent as compared with the above-mentioned human ALT; and thus have completed the present invention.

After studying from various directions a system that allows most effective expression of the altered-type human ALT, the present inventors have first paid attention on the utilization of the previous human ALT gene, and have carried out researches with reference to the amino acid sequence of native human ALT reported by Ishiguro et al. (Biochemistry, 30, 10451-10457, 1991), and finally, have paid attention on five amino acids in the human ALT gene for the first time.

Further, they have confirmed a combination that allows expression of the desired altered-type ALT by replacing codons for these five amino acids, and have newly developed a method that allows effective performance of this replacement, and have first succeeded to develop a total system for industrial production of the altered-type ALT.

Thus, they replaced the codons for the five amino acids in the human ALT gene based on the amino acid sequence of the native human ALT reported by Ishiguro et al., and simultaneously considered about an altered-type human ALT gene having restriction sites added at the upstream and downstream of said gene, a recombinant plasmid in which the altered-type human ALT gene is ligated to a vector plasmid, Escherichia coli transformed with said plasmid, and a process for production in which the human ALT is expressed as an active enzyme using the transformed Escherichia coli.

First, four kinds of gene fragments were amplified by means of PCR (Polymerase Chain Reaction) using a plasmid containing a cloned gene encoding the human ALT as a template and using eight kinds of primers for replacing the codons for the five amino acids in the human ALT gene. An altered human ALT gene (FIG. 1, FIG. 2 and FIG. 3: SEQ ID NO:1 in SEQUENCE LISTING) was obtained by exchanging a region of human ALT gene with the gene fragments amplified by said PCR, in order to replace the amino acids. Then a plasmid in which the altered-type human ALT gene was introduced was obtained, and Escherichia coli transformed by the recombinant plasmid was created. The human ALT was actually expressed as an active enzyme by culturing the transformed Escherichia coli, and thus the present invention was completed.

Accordingly, in the present invention is taken a DNA shown by Sequence No. 1 in Sequence Listing as the fundamental technical idea, and more specifically, are obtained a recombinant plasmid formed by replacing the codons for the five specific amino acids in the previously mentioned human ALT gene, preparing concurrently an ALT gene having restriction sites added at the upstream and downstream of said gene and ligating it to a vector plasmid, and Escherichia coli transformed with the recombinant plasmid formed by replacing the codons for the five amino acids in the human ALT gene, preparing concurrently an ALT gene having restriction sites added at the upstream and downstream of said gene and ligating it to a vector plasmid, and is collected ALT by culturing the transformant.

The plasmid of the present invention can be prepared, for example, by digesting the human ALT gene, and a DNA having a role as a promoter and a vector with restriction enzymes by the method described in J. Mol. Biol., 96, 171-184, 1974, and then ligating with a ligase, according to the method described in Biochem. Biophys. Acta, 72, 619-629, 1963.

The DNA having a role as a vector includes, for example, DNA's such as pBR 322 derived from Escherichia coli and the like. The promoter includes, for example, Tac promoter, tryptophan promoter, lambda PL promoter, lambda PR promoter, lactose promoter, T7 promoter and the like. The restriction enzyme includes, for example, EcoRI and BamHI and the ligase includes, for example, T4 DNA ligase.

The altered-type human ALT gene used in the present invention can be formed by amplifying four kinds of gene fragments by means of PCR using a cloned ALT gene derived from the human liver as a template and using eight kinds of primers for replacing the codons for the five amino acids in the human ALT gene, and exchanging a region of human ALT gene with the gene fragments amplified by said PCR, in order to replace the amino acids. Said eight kinds of primers used here are nucleotide sequences designed for replacing the codons for the five amino acids in the human ALT gene and for adding restriction sites at the upstream and downstream of the gene. Recombinant plasmid pTRAL-F140 for production of human ALT in the cells of Escherichia coli can be prepared by introducing the altered-type human ALT gene into expression vector pTRP. A transformant that produces the human ALT in the microbial cells can be obtained by transforming Escherichia coli with the recombinant plasmid having the altered-type human ALT gene of the present invention.

The cloned ALT gene derived from the human liver described above includes, for example, plasmid pHGT-39 (FIG. 5). In addition, as the primer, the sequences (FIG. 4), for example, can be used.

By culturing the transformant of Escherichia coli prepared in the above described manner under suitable medium conditions, it is possible to produce the human ALT, particularly the active human ALT having a sufficient enzyme activity in a large quantity. Isolation of the human ALT after cultivation can be performed, for example, by disrupting of the cells with a disrupting means such as ultra-sonication or the like, separation and purification.

Utilization of other host vector system, such as those of Bacillus subtilis, yeast, Chinese hamster oocyte (CHO) and the like, than the host vector system of Escherichia coli is also possible and the mass production of the human ALT can be conducted using these system.

EXAMPLES

The present invention will now be specifically described by means of Examples.

EXAMPLE 1

(a) Amplification of Partial Fragments of an Altered-type Human ALT Gene by PCR

Eight kinds of primers shown in FIG. 4, i.e. PF4-11, PFA-1 (SEQ ID NO:3) (SEQ ID NO:5), PF11 (SEQ ID NO:7), PFA-11 (SEQ ID NO:8), PF21 (SEQ ID NO:9), PFA-21 (SEQ ID NO:10), PF31(SEQ ID NO:12) and PFA-41 (SEQ ID NO:13), were obtained by DNA-synthesizing and four kinds of partial gene fragments were amplified by PCR with PF4-11 and PFA-1, PF11 and PFA-11, -PF21 and PFA-21, and PF31 and PFA-41, respectively, using a pHGT-39 containing a cloned human ALT gene as a template.

Of the above described primers, PF4-11, PF11, PF21 and PF31 were sense primers, PFA-1, PFA-11,. PFA-21 and PFA-41 were antisense primers, and they were designed on the basis of the nucleotide sequence of the human ALT. In the row above PF4-11 (SEQ ID NO:4), and the rows under PFA-1 (SEQ ID NO:6) and PFA-21 (SEQ ID NO:11), amino acid sequences encoded by them are shown. The underlines in nucleotide sequences of primers indicate the replaced bases and added restriction sites. The underlines in the amino acid sequence indicate the amino acid residues replaced by the replaced bases. Thus, primers were prepared according to a nucleotide sequence in which the fourth amino acid codon from the initiation codon for methionine (Met) in the amino acid sequence was replaced by a codon for serine (Ser), the fifth by a codon for threonine (Thr), the seventh by a codon for aspartic acid (Asp), the 39th by a codon for glycine (Gly) and the 222nd by a codon for alanine (Ala), based on the amino acid sequence of the native human ALT reported by Ishiguro et al. The reaction composition for amplification by PCR contained 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl₂, 0.01% (W/V) gelatin, each 100 μM of dNTP, each 0.5 μM of primers, 40 pg of the plasmid (pHGT-39) having the cloned human ALT gene, 100 μl containing 2.5 U of Taq polymerase (manufactured by Perkin Elmer Cetus) and 100 μl of mineral oil to be overlaid.

The reaction conditions included a denaturation step at 94° C. for 1 minute, an annealing step at 55° C. for 1 minute, and a polymerase reaction step at 72° C. for 1 minute and 15 seconds. This incubation was carried out at 35 cycles.

After contaminating protein in the reaction solution was removed by phenol extraction, the DNA fraction containing the PCR amplification products was precipitated by cold ethanol and recovered. Further, the four kinds of the partial fragments of the ALT gene were confirmed by agarose electrophoresis.

(b) Construction of a Human ALT Gene and Preparation of a Recombinant Plasmid pTRAL for the Purpose of its Expression

As an expression vector, pTRP of about 2.9 kb having a tryptophan promoter, Shine-Dalgarno sequence (SD sequence), and a multi-cloning site containing ECORI and BamHI was used.

Each 1 μg of gene fragments (PF4-11/PFA-1, PF11/PFA-11, PF21/PFA-21, and PF31/PFA-41) amplified by PCR were digested with combinations of restriction enzymes EcoRI-ApaI, ApaI-HincII, EcoRI-StuI and Eco47III-BamHI (all the restriction enzymes and enzyme reaction solutions theref or were manufactured by Takara Shuzo), respectively, at 37° C. for 2 hours. Respective reaction solutions (100 μl) were subjected to agarose electrophoresis, and the gel sections containing the digested gene partial fragments were excised out, melted at 65° C. and subjected to ethanol precipitation to recover the respective gene partial fragments (123 bp, 153 bp, 385 bp and 822 bp, respectively). Two gene partial fragments, i. e. PF4-11/PFA-1 and PF11/PFA-11, were mixed with pHGT-39 (4.4 kb) digested in advance with EcoRI-HincII and ligated with a DNA ligation kit (manufactured by Takara Shuzo) to give recombinant plasmid pHGT-F110 of 4.7 kb.

Two gene fragments, i. e. PF21/PFA-21 and PF31/PFA-41, were concurrently introduced into cloning vector PTZ18U (manufactured by Pharmacia) digested in advance with EcoRI-BamHI and subcloned. A partial fragment (1.1 kb) of the ALT gene formed by digesting the obtained plasmid with EcoO109I-BamHI and a partial fragment (393 bp) of the ALT gene formed by digesting pHGT-F110 with restriction enzymes EcoRI-EcoO109I were mixed with an expression vector pTRP (2.9 kb) digested with EcoRI-BamHI, and ligated with the same ligation kit to give recombinant plasmid pTRAL-F140 (4.4 kb) containing the human ALT gene, in which five amino acids were replaced. Then, Escherichia coli MV 1190 strain was transformed with this reaction solution according to the TSS (Transformation Storage Solution) method (Proc. Natl. Acad. Sci. USA, 86, 2172-2175, 1989). An Ampicillin resistant strain was cultured that emerged in LB agar medium (a medium (pH 7.4) prepared by dissolving 10 g of tryptone (manufactured by Difco), 5 g of yeast extract (manufactured by Difco), 10 g of NaCl and 15 g of powdered agar in 11 of distilled water) containing 50 μg/ml of Ampicillin. A plasmid DNA was prepared by the method of Bimboim, Dolyetal (NucleicAcids Res., 7, 1513-1523, 1979), cut with EcoRI and BamHI and then the fact was confirmed by agarose gel electrophoresis that 1.5 kb altered-type human ALT gene was correctly inserted into the expression vector pTRP. The process for preparing recombinant plasmid pTRAL-F140 is shown in FIG. 5, and the structure of the obtained plasmid pTRAL-F140 is shown in FIG. 6. The structure of expression vector pTRP is shown in FIG. 7, and the total nucleotide sequence thereof is shown in FIG. 8 and FIG. 9.

(c) Expression of the Recombinant Active Human ALT in Escherichia coli

Recombinant plasmid pTRAL-F140 prepared by the process shown in (b) was introduced into Escherichia coli MV1190 strain according to the TSS method and the analysis of the human ALT expressed by the obtained transformed Escherichia coli MV1190 (pTRAL-F140) was preformed as follows. The obtained transformed microbe was named Escherichia coli MV1190 (pTRAL-F140) and deposited under the Budapest Treaty in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, Japan, under an accession number FERM BP-5781 as the international deposit on January 8, 1997.

Transformed Escherichia coli MV1190 (pTRAL-F140) (FERM BP-5781) was inoculated in 200 ml of LB liquid medium, and after 18 hours at 30° C., when the culture reached the late stage of logarithmic growth phase, the microbe was transferred to 14 l of LB liquid medium. Similarly, after 18 hours at 30° C., when the culture reached the late stage of logarithmic growth phase, the cells were collected by centrifugation (10,000 ×g, 10 minutes). In 135 ml of Buffer A (40 mM acetate buffer (pH 5.5) containing 1 mM DTT, 1 mM EDTA, 5 mM 2-oxoglutarate and 50 μM pyridoxal phosphate) was suspended 27 g of the obtained wet cells. All the following operations were carried out at 4° C. After disrupting the cells by ultra-sonication, a cell-free extract was prepared by centrifugation (10,000 ×g, 10 minutes)

The cell-free extract was assayed for ALT activity and it was found that it has an activity of 1.58 U/mg protein. On the other hand, no ALT activity was found in the case of Escherichia coli host MV 1190, and accordingly, it could be confirmed that the activity detected in the transformed MV 1190 (pTRAL-F140) was derived from the recombinant human ALT.

Further, to 10 μl of this cell-free extract was added two times the volume of a sample treating solution (50 mM Tris-HCl (pH 6.8), 6% SDS, 20% glycerol, 200 mM dithiothreitol and 3mM phenylmethanesulfonyl fluoride (PMSF)). The mixture was heated at 60° C. for 30 minutes and subjected to SDS-polyacrylamide gel electrophoresis according to the method of Laemmli et al (Nature, 227, 680-685, 1970). After migration, the gel was stained with Coomassie Brilliant Blue R-250. As the result, a band of the human ALT at a molecular weight of about 55 k was detected, and further, this protein band showed a specific cross-reaction with anti-human ALT antibody. Accordingly, it was confirmed that the recombinant Escherichia coli MV 1190 (pTRAL-F140) effectively expressed the transformed human ALT as an active enzyme.

(d) Purification of the Recombinant Active Human ALT

In 135 ml of Buffer A (40 mM acetate buffer (pH 5.5) containing 1 mM DTT, 1 mM EDTA, 5 mM 2-oxoglutarate and 50 μM pyridoxal phosphate) was suspended 27 g of the wet cells of MV 1190 (pTRAL-F140). All the following operations were carried out at 4° C. After disrupting the cells by ultra-sonication, precipitate was removed by centrifugation (10,000 ×g, 10 minutes) to give a cell-free extract. To the extract (145 ml) was added 16.5 g of ammonium sulfate (20% saturation) and the mixture was stirred for 1.5 hour. After centrifugation (10,000 ×g, 10 minutes), 27.8 g of ammonium sulfate (50% saturation) was added to the obtained supernatant (147 ml) and the mixture was stirred for 1 hour. Precipitate under 20-50% saturation containing ALT activity was recovered by centrifugation (10,000 ×g, 10 minutes). In 33.4 ml of Buffer A was suspended the precipitate and the mixture was dialyzed against the same buffer for 15hours. After bringing the volume to 83.5 ml by addition of Buffer A, 12 g of ammonium sulfate (25% saturation) was added to the mixture, which was stirred for 1 hour and centrifuged (10,000 ×g, 10 minutes) to give a supernatant. The supernatant was applied onto Butyl-Toyopearl 650M (2.6×30 cm) (manufactured by Tosoh) equilibrated with Buffer Acontainingl.09Mammoniumsulfate. After washing with Buffer A containing 1.09 M ammonium sulfate, the recombinant h-ALT (human ALT) was eluted with a linear gradient of 1.09 M - 0 M ammonium sulfate.

The obtained active fraction was dialyzed against Buffer A for 15 hours and then applied onto CM-Sepharose CL6B (2.6×5 cm) (manufactured by Pharmacia) equilibrated with Buffer A. After washing with Buffer A containing 100 mM NaCl, the ALT was eluted with a linear gradient of 100-200 mM NaCl to give 5.45 mg protein of purified recombinant h-ALT. Its specific activity was 277 U/mg protein. As the result of SDS-polyacrylamide gel electrophoresis conducted with 1 μg of the purified preparation and staining with Coomassie Brilliant Blue R-250, only a band corresponding to the recombinant human ALT was confirmed.

(e) Comparison of the Obtained Human ALT

Properties of the recombinant active human ALT (altered ALT) obtained in this manner and those of non-altered ALT were compared as follows.

i) Difference in Translated Amino Acid Sequences

Five amino acid residues were different before and after the alteration. The amino acid sequence translated from the ALT gene after alteration was identical to the amino acid sequence, analyzed with a protein sequencer, of the native ALT purified from the human liver by Ishiguro et al. (Ishiguro et al., (1991), Biochemistry, 30, 10451-10457).

ii) Difference in Specific Activity

As described above, it was confirmed that the recombinant Escherichia coli MV 1190 (pTRAL-F140) effectively expressed the recombinant human ALT as an active enzyme. Although the amount of expression as a protein was not different before and after the alteration, the ALT activity in the crude cell extract was 1.58 U/mg protein after alteration as compared with 0.174 U/mg protein before alteration. When the comparison was made on the purified preparations, the activity was 277 U/mg protein after alteration as compared with 34.5 U/mg protein before alteration. Thus, it was confirmed that the ALT after alteration had a higher specific activity.

iii) Difference in Km Value

L-alanine 2-oxoglutaric acid Before alteration 83.3 mM  6.99 mM After alteration 20.5 mM 0.443 mM

While the ALT before alteration had Km values for L-alanine and 2-oxoglutaric acid as the substrates quite different from those of the native ALT, the ALT after alteration had the same values as those of the native ALT.

iv) Difference in Isoelectric Point

When isoelectric point (pI) of ALT was measured by conducting electric focusing, it was found that the ALT before alteration had a pI of about 7.6 and the ALT after alteration had a pI of about 6.5. These values of isoelectric point roughly agreed with values estimated from the amino acid sequences. The pI value for the ALT after alteration was close to the pI value for the native ALT reported by Kanemitsu et al. (Kanemitsu, F. et al., Clin. Biochem., 23, 121-125, 1990).

By altering the human ALT gene to a nucleotide sequence encoding an amino acid sequence in which five amino acid residues are replaced through PCR and putting the altered gene under control of tryptophan promoter, the recombinant plasmid of the present invention can effectively express the recombinant human ALT as an active enzyme in the cells of Escherichia coli. Further, the obtained recombinant human ALT is the same as the naturally occurring ALT derived from the human liver and can be utilized, after purification, as the standard sample in serum diagnosis for exactly determining the amount of ALT leaked into serum in liver diseases.

Reference to Deposited Microorganism under Rule 13, bis

1. Escherichia coli MV1190 (pTRAL-F140)

A. Name and address of the depository institution in which said microorganism has been deposited:

Name: National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry

Address: 1-3, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken 305, Japan

B. Date of deposition in the depository institution in A. Jan. 8, 1997

C. Accession number given to the deposit by the depository institution in A.

FERM BP-5781

14 1 1505 DNA Homo sapiens CDS (9)..(1496) 1 gaattcat atg gca agc tca aca ggt gat aga tct cag gcg gtg agg cat 50 Met Ala Ser Ser Thr Gly Asp Arg Ser Gln Ala Val Arg His 1 5 10 gga ctg agg gcg aag gtg ctg acg ctg gac ggc atg aac ccg cgt gtg 98 Gly Leu Arg Ala Lys Val Leu Thr Leu Asp Gly Met Asn Pro Arg Val 15 20 25 30 cgg aga gtg gag tac gca gtg cgt ggg ccc ata gtg cag cga gcc ttg 146 Arg Arg Val Glu Tyr Ala Val Arg Gly Pro Ile Val Gln Arg Ala Leu 35 40 45 gag ctg gag cag gag ctg cgc cag ggt gtg aag aag cct ttc acc gag 194 Glu Leu Glu Gln Glu Leu Arg Gln Gly Val Lys Lys Pro Phe Thr Glu 50 55 60 gtc atc cgt gcc aac atc ggg gac gca cag gct atg ggg cag agg ccc 242 Val Ile Arg Ala Asn Ile Gly Asp Ala Gln Ala Met Gly Gln Arg Pro 65 70 75 atc acc ttc ctg cgc cag gtc ttg gcc ctc tgt gtt aac cct gat ctt 290 Ile Thr Phe Leu Arg Gln Val Leu Ala Leu Cys Val Asn Pro Asp Leu 80 85 90 ctg agc agc ccc aac ttc cct gac gat gcc aag aaa agg gcg gag cgc 338 Leu Ser Ser Pro Asn Phe Pro Asp Asp Ala Lys Lys Arg Ala Glu Arg 95 100 105 110 atc ttg cag gcg tgt ggg ggc cac agt ctg ggg gcc tac agc gtc agc 386 Ile Leu Gln Ala Cys Gly Gly His Ser Leu Gly Ala Tyr Ser Val Ser 115 120 125 tcc ggc atc cag ctg atc cgg gag gac gtg gcg cgg tac att gag agg 434 Ser Gly Ile Gln Leu Ile Arg Glu Asp Val Ala Arg Tyr Ile Glu Arg 130 135 140 cgt gac gga ggc atc cct gcg gac ccc aac aac gtc ttc ctg tcc aca 482 Arg Asp Gly Gly Ile Pro Ala Asp Pro Asn Asn Val Phe Leu Ser Thr 145 150 155 ggg gcc agc gat gcc atc gtg acg gtg ctg aag ctg ctg gtg gcc ggc 530 Gly Ala Ser Asp Ala Ile Val Thr Val Leu Lys Leu Leu Val Ala Gly 160 165 170 gag ggc cac aca cgc acg ggt gtg ctc atc ccc atc ccc cag tac cca 578 Glu Gly His Thr Arg Thr Gly Val Leu Ile Pro Ile Pro Gln Tyr Pro 175 180 185 190 ctc tac tcg gcc acg ctg gca gag ctg ggc gca gtg cag gtg gat tac 626 Leu Tyr Ser Ala Thr Leu Ala Glu Leu Gly Ala Val Gln Val Asp Tyr 195 200 205 tac ctg gac gag gag cgt gcc tgg gcg ctg gac gtg gcc gag ctt gct 674 Tyr Leu Asp Glu Glu Arg Ala Trp Ala Leu Asp Val Ala Glu Leu Ala 210 215 220 agg gct ctg ggc cag gcg cgt gac cac tgc cgc cct cgt gcg ctc tgt 722 Arg Ala Leu Gly Gln Ala Arg Asp His Cys Arg Pro Arg Ala Leu Cys 225 230 235 gtc atc aac cct ggc aac ccc acc ggg cag gtg cag acc cgc gag tgc 770 Val Ile Asn Pro Gly Asn Pro Thr Gly Gln Val Gln Thr Arg Glu Cys 240 245 250 atc gag gcc gtg atc cgc ttc gcc ttc gaa gag cgg ctc ttt ctg ctg 818 Ile Glu Ala Val Ile Arg Phe Ala Phe Glu Glu Arg Leu Phe Leu Leu 255 260 265 270 gcg gac gag gtg tac cag gac aac gtg tac gcc gcg ggt tcg cag ttc 866 Ala Asp Glu Val Tyr Gln Asp Asn Val Tyr Ala Ala Gly Ser Gln Phe 275 280 285 cac tca ttc aag aag gtg ctc atg gag atg ggg ccg ccc tac gcc ggg 914 His Ser Phe Lys Lys Val Leu Met Glu Met Gly Pro Pro Tyr Ala Gly 290 295 300 cag cag gag ctt gcc tcc ttc cac tcc acc tcc aaa ggc tac atg ggc 962 Gln Gln Glu Leu Ala Ser Phe His Ser Thr Ser Lys Gly Tyr Met Gly 305 310 315 gag tgc ggg ttc cgc ggc ggc tat gtg gag gtg gtg aac atg gac gct 1010 Glu Cys Gly Phe Arg Gly Gly Tyr Val Glu Val Val Asn Met Asp Ala 320 325 330 gca gtg cag cag cag atg ctg aag ctg atg agt gtg cgg ctg tgc ccg 1058 Ala Val Gln Gln Gln Met Leu Lys Leu Met Ser Val Arg Leu Cys Pro 335 340 345 350 ccg gtg cca gga cag gcc ctg ctg gac ctg gtg gtc agc ccg ccc gcg 1106 Pro Val Pro Gly Gln Ala Leu Leu Asp Leu Val Val Ser Pro Pro Ala 355 360 365 ccc acc gac ccc tcc ttt gcg cag ttc cag gct gag aag cag gca gtg 1154 Pro Thr Asp Pro Ser Phe Ala Gln Phe Gln Ala Glu Lys Gln Ala Val 370 375 380 ctg gca gag ctg gcg gcc aag gcc aag ctc acc gag cag gtc ttc aat 1202 Leu Ala Glu Leu Ala Ala Lys Ala Lys Leu Thr Glu Gln Val Phe Asn 385 390 395 gag gct cct ggc atc agc tgc aac cca gtg cag ggc gcc atg tac tcc 1250 Glu Ala Pro Gly Ile Ser Cys Asn Pro Val Gln Gly Ala Met Tyr Ser 400 405 410 ttc ccg cgc gtg cag ctg ccc ccg cgg gcg gtg gag cgc gct cag gag 1298 Phe Pro Arg Val Gln Leu Pro Pro Arg Ala Val Glu Arg Ala Gln Glu 415 420 425 430 ctg ggc ctg gcc ccc gat atg ttc ttc tgc ctg cgc ctc ctg gag gag 1346 Leu Gly Leu Ala Pro Asp Met Phe Phe Cys Leu Arg Leu Leu Glu Glu 435 440 445 acc ggc atc tgc gtg gtg cca ggg agc ggc ttt ggg cag cgg gaa ggc 1394 Thr Gly Ile Cys Val Val Pro Gly Ser Gly Phe Gly Gln Arg Glu Gly 450 455 460 acc tac cac ttc cgg atg acc att ctg ccc ccc ttg gag aaa ctg cgg 1442 Thr Tyr His Phe Arg Met Thr Ile Leu Pro Pro Leu Glu Lys Leu Arg 465 470 475 ctg ctg ctg gag aag ctg agc agg ttc cat gcc aag ttc acc ctc gag 1490 Leu Leu Leu Glu Lys Leu Ser Arg Phe His Ala Lys Phe Thr Leu Glu 480 485 490 tac tcc tgaggatcc 1505 Tyr Ser 495 2 496 PRT Homo sapiens 2 Met Ala Ser Ser Thr Gly Asp Arg Ser Gln Ala Val Arg His Gly Leu 1 5 10 15 Arg Ala Lys Val Leu Thr Leu Asp Gly Met Asn Pro Arg Val Arg Arg 20 25 30 Val Glu Tyr Ala Val Arg Gly Pro Ile Val Gln Arg Ala Leu Glu Leu 35 40 45 Glu Gln Glu Leu Arg Gln Gly Val Lys Lys Pro Phe Thr Glu Val Ile 50 55 60 Arg Ala Asn Ile Gly Asp Ala Gln Ala Met Gly Gln Arg Pro Ile Thr 65 70 75 80 Phe Leu Arg Gln Val Leu Ala Leu Cys Val Asn Pro Asp Leu Leu Ser 85 90 95 Ser Pro Asn Phe Pro Asp Asp Ala Lys Lys Arg Ala Glu Arg Ile Leu 100 105 110 Gln Ala Cys Gly Gly His Ser Leu Gly Ala Tyr Ser Val Ser Ser Gly 115 120 125 Ile Gln Leu Ile Arg Glu Asp Val Ala Arg Tyr Ile Glu Arg Arg Asp 130 135 140 Gly Gly Ile Pro Ala Asp Pro Asn Asn Val Phe Leu Ser Thr Gly Ala 145 150 155 160 Ser Asp Ala Ile Val Thr Val Leu Lys Leu Leu Val Ala Gly Glu Gly 165 170 175 His Thr Arg Thr Gly Val Leu Ile Pro Ile Pro Gln Tyr Pro Leu Tyr 180 185 190 Ser Ala Thr Leu Ala Glu Leu Gly Ala Val Gln Val Asp Tyr Tyr Leu 195 200 205 Asp Glu Glu Arg Ala Trp Ala Leu Asp Val Ala Glu Leu Ala Arg Ala 210 215 220 Leu Gly Gln Ala Arg Asp His Cys Arg Pro Arg Ala Leu Cys Val Ile 225 230 235 240 Asn Pro Gly Asn Pro Thr Gly Gln Val Gln Thr Arg Glu Cys Ile Glu 245 250 255 Ala Val Ile Arg Phe Ala Phe Glu Glu Arg Leu Phe Leu Leu Ala Asp 260 265 270 Glu Val Tyr Gln Asp Asn Val Tyr Ala Ala Gly Ser Gln Phe His Ser 275 280 285 Phe Lys Lys Val Leu Met Glu Met Gly Pro Pro Tyr Ala Gly Gln Gln 290 295 300 Glu Leu Ala Ser Phe His Ser Thr Ser Lys Gly Tyr Met Gly Glu Cys 305 310 315 320 Gly Phe Arg Gly Gly Tyr Val Glu Val Val Asn Met Asp Ala Ala Val 325 330 335 Gln Gln Gln Met Leu Lys Leu Met Ser Val Arg Leu Cys Pro Pro Val 340 345 350 Pro Gly Gln Ala Leu Leu Asp Leu Val Val Ser Pro Pro Ala Pro Thr 355 360 365 Asp Pro Ser Phe Ala Gln Phe Gln Ala Glu Lys Gln Ala Val Leu Ala 370 375 380 Glu Leu Ala Ala Lys Ala Lys Leu Thr Glu Gln Val Phe Asn Glu Ala 385 390 395 400 Pro Gly Ile Ser Cys Asn Pro Val Gln Gly Ala Met Tyr Ser Phe Pro 405 410 415 Arg Val Gln Leu Pro Pro Arg Ala Val Glu Arg Ala Gln Glu Leu Gly 420 425 430 Leu Ala Pro Asp Met Phe Phe Cys Leu Arg Leu Leu Glu Glu Thr Gly 435 440 445 Ile Cys Val Val Pro Gly Ser Gly Phe Gly Gln Arg Glu Gly Thr Tyr 450 455 460 His Phe Arg Met Thr Ile Leu Pro Pro Leu Glu Lys Leu Arg Leu Leu 465 470 475 480 Leu Glu Lys Leu Ser Arg Phe His Ala Lys Phe Thr Leu Glu Tyr Ser 485 490 495 3 54 DNA Homo sapiens CDS (11)..(52) 3 gggaattcat atg gca agc tca aca ggt gat aga tct cag gcg gtg agg 49 Met Ala Ser Ser Thr Gly Asp Arg Ser Gln Ala Val Arg 1 5 10 cat gg 54 His 4 14 PRT Homo sapiens 4 Met Ala Ser Ser Thr Gly Asp Arg Ser Gln Ala Val Arg His 1 5 10 5 26 DNA Homo sapiens 5 tatgggccca cgcactgcgt actcca 26 6 8 PRT Homo sapiens 6 Glu Tyr Ala Val Arg Gly Pro Ile 1 5 7 27 DNA Homo sapiens 7 cgtgggccca tagtgcagcg agccttg 27 8 22 DNA Homo sapiens 8 ttggcatcgt cagggaagtt gg 22 9 30 DNA Homo sapiens 9 ctggaattcc ccaacttccc tgacgatgcc 30 10 33 DNA Homo sapiens 10 cagaggccta gcaagctcgg ccacgtccag cgc 33 11 9 PRT Homo sapiens 11 Ala Leu Asp Val Ala Glu Leu Ala Arg 1 5 12 27 DNA Homo sapiens 12 cacagcgctc tgggccaggc gcgtgac 27 13 42 DNA Homo sapiens 13 ggggatcctc aggagtactc gagggtgaac ttggcatgga ac 42 14 2927 DNA Artificial Sequence Description of Artificial Sequence Hybrid of human and artificial vector sequences 14 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctct 240 aatacgactc actataggga aagcttccct gttgacaatt aatcatcgaa ctagttaaca 300 gtacgcaagt tcacgtaaaa agggtagaat tcgagctcgg tacccgggga tcctctagag 360 tcgacctgca ggtcgaaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 420 tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 480 cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga 540 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 600 tacacttgcc agcgccctac cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 660 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 720 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 780 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 840 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 900 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 960 cgcgaatttt aacaaaatat taacgtttac aatttcaggt ggcacttttc ggggaaatgt 1020 gcccggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag 1080 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca 1140 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc 1200 agaaacgctg gtgaaactaa aagatgctga agatcagttg ggtgcacgag tgggttacat 1260 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc 1320 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg 1380 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc 1440 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat 1500 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga 1560 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc 1620 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc 1680 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt 1740 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc 1800 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc 1860 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca 1920 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca 1980 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt 2040 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta 2100 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg 2160 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc 2220 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag 2280 cagagcgcag ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa 2340 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc 2400 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc 2460 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta 2520 caccgaactg agatacctac agcgtgagca ttgagaaagc gccacgcttc ccgaagggag 2580 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct 2640 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga 2700 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc 2760 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 2820 atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 2880 cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaag 2927 

What is claimed is:
 1. An isolated DNA molecule, comprising the nucleotide sequence from nucleotides 9 to 1499 of SEQ ID NO:1.
 2. The isolated DNA molecule according to claim 1, wherein restriction sites are linked to the sequence of nucleotides 9 to 1499 of SEQ ID NO:1 at the initiation end and the terminal end thereof.
 3. The isolated DNA molecule according to claim 1, which comprises the nucleotide sequence from nucleotides 1 to 1505 of SEQ ID NO:1.
 4. A recombinant plasmid in which the DNA molecule according to claim 2 is inserted into a vector plasmid.
 5. An Escherichia coli host cell transformed with the recombinant plasmid of claim
 4. 6. A process for producing an active human alanine aminotransferase (ALT), comprising: cultivating the transformed Escherichia coli host cell of claim 5 in a culture medium; and recovering ALT from the resultant cells thereof.
 7. A recombinant plasmid in which the DNA molecule according to claim 3 is inserted into a vector plasmid.
 8. An Escherichia coli host cell transformed with the recombinant plasmid according to claim
 7. 9. A process for producing an active alanine aminotransferase (ALT), comprising: cultivating the transformed Escherichia coli host cell of claim 8 in a culture medium; and recovering ALT from the resultant cells thereof.
 10. Recombinant plasmid pTRAL-F140.
 11. An Escherichia coli host cell transformed with the recombinant plasmid of claim
 10. 12. A process for producing an active alanine aminotransferase (ALT), comprising: cultivating the transformed Escherichia coli host cell of claim 11 in a culture medium; and recovering ALT from the resultant cells thereof.
 13. A biologically pure culture of Escherichia coli MV1190 (pTRAL-Fl40), FERM BP-5781.
 14. A process for producing an active alanine aminotransferase (ALT), comprising: cultivating Escherichia coli MV1190 (pTRAL-Fl40), FERM BP-5781, of claim 13 in a culture medium; and recovering ALT from the resultant cells thereof. 